Our recent genome-scale survey of genes essential in ovarian cancer cell lines coupled with the comprehensive characterization of ovarian cancer genomes pinpointed the expression of the PAX8 transcription factor as a critical vulnerability of the majority of ovarian tumors. PAX8 is a member of the paired- box family of transcription factors expressed during the development of the female Mllerian ducts and in the fallopian tube, which is considered the origin of ovarian carcinomas. PAX8 is overexpressed or amplified in most ovarian tumors or cell lines indicating that many ovarian cancer cells have co-opted PAX8 transcriptional program to maintain their survival and proliferation. We found that PAX8 was the highest ranked gene required for the survival of ovarian cancer cell lines but not normal ovarian cells or cells from a variety of other lineages. Characterizing the underlying biological mechanisms behind this novel dependency of ovarian tumors requires PAX8-specific chemical probes, however such probes have not been reported to date. This project focuses on the identification of small-molecule inhibitors of PAX8 activity that specifically inhibit the survivalof ovarian cancer cells expressing PAX8. Such inhibitors will not only facilitate the investigation of the biological mechanism behind the addiction of ovarian cancers to PAX8 but may also serve as useful starting points for future therapeutic avenues of development. To identify inhibitors of PAX8 activity we have developed and validated a cell-based Luc reporter gene assay in 384-well format that measures PAX8 activity in ovarian cancer cells. We selected an ovarian cancer cell line (from a panel of 25 well-characterized ovarian cancer cell lines) that expresses high levels of PAX8 and is strongly dependent on PAX8 expression, and thus is highly relevant to PAX8 biology. Compounds that suppress PAX8 activity will be subjected to two counter screens to remove: i) non-specific inhibitors of transcription; ii) Luc inhibitors; and iii) generally cytooxic compounds. We wil then characterize compounds that preferentially reduce the survival of PAX8-dependent versus independent cancer cells. Top scoring compounds will then be optimized to achieve the desired selectivity, efficacy and potency. As a short-term goal, we will use these compounds as probes to elucidate the molecular basis of PAX8 essentiality in ovarian tumors. In parallel, the direct binding targets will be identified using mass-spectrometry- based proteomics, providing important knowledge on upstream regulators and downstream effectors of PAX8. Since our preliminary work suggests that PAX8 expression represents a clear biomarker that predicts sensitivity of ovarian tumors to PAX8 suppression, we envision using these PAX8 modulators in xenograft models of ovarian cancer as we work towards a longer-term goal of translating these findings into therapeutic opportunities.